Keith E.J. Tyo

Terpenoids: opportunities for biosynthesis of natural product drugs using engineered microorganisms.

Terpenoids represent a diverse class of molecules that provide a wealth of opportunities to address many human health and societal issues. The expansive array of structures and functionalities that have been evolved in nature provide an excellent pool of molecules for use in human therapeutics. While this class of molecules has members with therapeutic properties including anticancer, antiparasitic, antimicrobial, antiallergenic, antispasmodic, antihyperglycemic, anti-inflammatory, and immunomodulatory properties, supply limitations prevent the large scale use of some molecules. Many of these molecules are only found in ppm levels in nature thus requiring massive harvesting to obtain sufficient amounts of the drug. Synthetic biology and metabolic engineering provide innovative approaches to increase the production of the desired molecule in the native organism, and most importantly, transfer the biosynthetic pathways to other hosts. Microbial systems are well studied, and genetic manipulations allow the optimization of microbial metabolisms for the production of common terpenoid precursors. Using a host of tools, unprecedented advancements in the large scale production of terpenoids have been achieved in recent years. Identification of limiting steps and pathway regulation, coupled with design strategies to minimize terpenoid byproducts wih a high flux to the desired biosynthetic pathways, have yielded greater than 100-fold improvements in the production of a range of terpenoids. This review focuses on the biodiversity of terpenoids, the biosynthetic pathways involved, and engineering efforts to maximize the production through these pathways.

Stabilized gene duplication enables long-term selection-free heterologous pathway expression

Engineering robust microbes for the biotech industry typically requires high-level, genetically stable expression of heterologous genes and pathways. Although plasmids have been used for this task, fundamental issues concerning their genetic stability have not been adequately addressed. Here we describe chemically inducible chromosomal evolution (CIChE), a plasmid-free, high gene copy expression system for engineering Escherichia coli. CIChE uses E. coli recA homologous recombination to evolve a chromosome with ∼40 consecutive copies of a recombinant pathway. Pathway copy number is stabilized by recA knockout, and the resulting engineered strain requires no selection markers and is unaffected by plasmid instabilities. Comparison of CIChE-engineered strains with equivalent plasmids revealed that CIChE improved genetic stability approximately tenfold and growth phase–specific productivity approximately fourfold for a strain producing the high metabolic burden–biopolymer poly-3-hydroxybutyrate. We also increased the yield of the nutraceutical lycopene by 60%. CIChE should be applicable in many organisms, as it only requires having targeted genomic integration methods and a recA homolog.

Metabolic engineering of recombinant protein secretion by Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a widely used cell factory for the production of fuels and chemicals, and it is also provides a platform for the production of many heterologous proteins of medical or industrial interest. Therefore, many studies have focused on metabolic engineering S. cerevisiae to improve the recombinant protein production, and with the development of systems biology, it is interesting to see how this approach can be applied both to gain further insight into protein production and secretion and to further engineer the cell for improved production of valuable proteins. In this review, the protein post-translational modification such as folding, trafficking, and secretion, steps that are traditionally studied in isolation will here be described in the context of the whole system of protein secretion. Furthermore, examples of engineering secretion pathways, high-throughput screening and systems biology applications of studying protein production and secretion are also given to show how the protein production can be improved by different approaches. The objective of the review is to describe individual biological processes in the context of the larger, complex protein synthesis network.

Toward Design-based Engineering of Industrial Microbes

Engineering industrial microbes has been hampered by incomplete knowledge of cell biology. Thus an iterative engineering cycle of modeling, implementation, and analysis has been used to increase knowledge of the underlying biology while achieving engineering goals. Recent advances in Systems Biology technologies have drastically improved the amount of information that can be collected in each iteration. As well, Synthetic Biology tools are melding modeling and molecular implementation. These advances promise to move microbial engineering from the iterative approach to a design-oriented paradigm, similar to electrical circuits and architectural design. Genome-scale metabolic models, new tools for controlling expression, and integrated -omics analysis are described as key contributors in moving the field toward Design-based Engineering.

High-Throughput Screen for Poly-3-Hydroxybutyrate in Escherichia coli and Synechocystis sp. Strain PCC6803

ABSTRACT A novel, quantitative method for detecting poly-3-hydroxybutyrate (PHB) amounts in viable cells was developed to allow for high-throughput screening of mutant libraries. The staining technique was demonstrated and optimized for the cyanobacterium Synechocystis sp. strain PCC6803 and the eubacterium Escherichia coli to maximize the fluorescence difference between PHB-accumulating and control cells by flow cytometry. In Synechocystis, the level of nonspecific dye binding was reduced by using nonionic stain buffer that allowed quantitation of fluorescence levels. In E. coli, the use of a mild sucrose shock facilitated uptake of Nile red without significant loss of viability. The optimized staining protocols yielded a linear response for the mean fluorescence against (chemically measured) PHB. The staining protocols are novel methods useful in the high-throughput evaluation of combinatorial libraries of Synechocystis and E. coli using fluorescence-activated cell sorting to identify mutants with increased PHB-accumulating properties.

MINEs: open access databases of computationally predicted enzyme promiscuity products for untargeted metabolomics

BackgroundIn spite of its great promise, metabolomics has proven difficult to execute in an untargeted and generalizable manner. Liquid chromatography–mass spectrometry (LC–MS) has made it possible to gather data on thousands of cellular metabolites. However, matching metabolites to their spectral features continues to be a bottleneck, meaning that much of the collected information remains uninterpreted and that new metabolites are seldom discovered in untargeted studies. These challenges require new approaches that consider compounds beyond those available in curated biochemistry databases.DescriptionHere we present Metabolic In silico Network Expansions (MINEs), an extension of known metabolite databases to include molecules that have not been observed, but are likely to occur based on known metabolites and common biochemical reactions. We utilize an algorithm called the Biochemical Network Integrated Computational Explorer (BNICE) and expert-curated reaction rules based on the Enzyme Commission classification system to propose the novel chemical structures and reactions that comprise MINE databases. Starting from the Kyoto Encyclopedia of Genes and Genomes (KEGG) COMPOUND database, the MINE contains over 571,000 compounds, of which 93% are not present in the PubChem database. However, these MINE compounds have on average higher structural similarity to natural products than compounds from KEGG or PubChem. MINE databases were able to propose annotations for 98.6% of a set of 667 MassBank spectra, 14% more than KEGG alone and equivalent to PubChem while returning far fewer candidates per spectra than PubChem (46 vs. 1715 median candidates). Application of MINEs to LC–MS accurate mass data enabled the identity of an unknown peak to be confidently predicted.ConclusionsMINE databases are freely accessible for non-commercial use via user-friendly web-tools at http://minedatabase.mcs.anl.gov and developer-friendly APIs. MINEs improve metabolomics peak identification as compared to general chemical databases whose results include irrelevant synthetic compounds. Furthermore, MINEs complement and expand on previous in silico generated compound databases that focus on human metabolism. We are actively developing the database; future versions of this resource will incorporate transformation rules for spontaneous chemical reactions and more advanced filtering and prioritization of candidate structures.

Engineering of vesicle trafficking improves heterologous protein secretion in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often restricted due to the limitations of the host strain. In the protein secretory pathway, the protein trafficking between different organelles is catalyzed by the soluble NSF (N-ethylmaleimide-sensitive factor) receptor (SNARE) complex and regulated by the Sec1/Munc18 (SM) proteins. In this study, we report that over-expression of the SM protein encoding genes SEC1 and SLY1, improves the protein secretion in S. cerevisiae. Engineering Sec1p, the SM protein that is involved in vesicle trafficking from Golgi to cell membrane, improves the secretion of heterologous proteins human insulin precursor and α-amylase, and also the secretion of an endogenous protein invertase. Enhancing Sly1p, the SM protein regulating the vesicle fusion from endoplasmic reticulum (ER) to Golgi, increases α-amylase production only. Our study demonstrates that strengthening the protein trafficking in ER-to-Golgi and Golgi-to-plasma membrane process is a novel secretory engineering strategy for improving heterologous protein production in S. cerevisiae.

Different expression systems for production of recombinant proteins in Saccharomyces cerevisiae

Yeast Saccharomyces cerevisiae has become an attractive cell factory for production of commodity and speciality chemicals and proteins, such as industrial enzymes and pharmaceutical proteins. Here we evaluate most important expression factors for recombinant protein secretion: we chose two different proteins (insulin precursor (IP) and α‐amylase), two different expression vectors (POTud plasmid and CPOTud plasmid) and two kinds of leader sequences (the glycosylated alpha factor leader and a synthetic leader with no glycosylation sites). We used IP and α‐amylase as representatives of a simple protein and a multi‐domain protein, as well as a non‐glycosylated protein and a glycosylated protein, respectively. The genes coding for the two recombinant proteins were fused independently with two different leader sequences and were expressed using two different plasmid systems, resulting in eight different strains that were evaluated by batch fermentations. The secretion level (µmol/L) of IP was found to be higher than that of α‐amylase for all expression systems and we also found larger variation in IP production for the different vectors. We also found that there is a change in protein production kinetics during the diauxic shift, that is, the IP was produced at higher rate during the glucose uptake phase, whereas amylase was produced at a higher rate in the ethanol uptake phase. For comparison, we also refer to data from another study, (Tyo et al. submitted) in which we used the p426GPD plasmid (standard vector using URA3 as marker gene and pGPD1 as expression promoter). For the IP there is more than 10‐fold higher protein production with the CPOTud vector compared with the standard URA3‐based vector, and this vector system therefore represent a valuable resource for future studies and optimization of recombinant protein production in yeast. Biotechnol. Bioeng. 2012; 109:1259–1268.

Analysis of polyhydroxybutyrate flux limitations by systematic genetic and metabolic perturbations

Poly-3-hydroxybutyrate (PHB) titers in Escherichia coli have benefited from 10+ years of metabolic engineering. In the majority of studies, PHB content, expressed as percent PHB (dry cell weight), is increased, although this increase can be explained by decreases in growth rate or increases in PHB flux. In this study, growth rate and PHB flux were quantified directly in response to systematic manipulation of (1) gene expression in the product-forming pathway and (2) growth rates in a nitrogen-limited chemostat. Gene expression manipulation revealed acetoacetyl-CoA reductase (phaB) limits flux to PHB, although overexpression of the entire pathway pushed the flux even higher. These increases in PHB flux are accompanied by decreases in growth rate, which can be explained by carbon diversion, rather than toxic effects of the PHB pathway. In chemostats, PHB flux was insensitive to growth rate. These results imply that PHB flux is primarily controlled by the expression levels of the product forming pathway and not by the availability of precursors. These results confirm prior in vitro measurements and metabolic models and show expression level is a major affecter of PHB flux.

Virus-like particles: the future of microbial factories and cell-free systems as platforms for vaccine development

Vaccines based on virus-like particles have proved their success in human health. More than 25 years after the approval of the first vaccine based on this technology, the substantial efforts to expand the range of applications and target diseases are beginning to bear fruit. The incursion of high-throughput screening technologies, combined with new developments in protein engineering and chemical coupling, have accelerated the development of systems capable of producing macrostructures useful for vaccinology, gene delivery, immunotherapy and bionanotechnology. This review summarizes the most recent developments in microbial cell factories and cell-free systems for virus-like particle production and discusses the future impact of this technology in human and animal health.